Gram-negative neonatal bacterial meningitis is a major cause of mortality and morbidity worldwide with neonatal meningitis Escherichia coli (NMEC) being the most common agent of these infections. Most of the survivors of Gram-negative neonatal bacterial meningitis suffer neurologic sequelae or developmental abnormalities, and if untreated, the disease is usually fatal. Like other extraintestinal pathogeni E. coli (ExPEC), NMEC are well equipped to survive outside the intestine and cause disease in the extraintestinal compartment. However, NMEC are distinguished from other ExPEC by their abilities to reach high counts in the bloodstream and cross the blood-brain barrier. Here, we present evidence that much of what we know about NMEC pathogenesis is based on atypical NMEC strains that lack one of NMEC's most common traits, their large putative virulence plasmids. Our research with very similar plasmids in avian pathogenic E. coli (APEC) and our preliminary studies of NMEC, suggest that this is an important oversight and have led us to hypothesize that NMEC plasmids have evolved to increase NMEC fitness and are critical to the pathogenesis of meningitis by contributing to bloodstream growth and blood-brain barrier traversal. To test this hypothesis, we will generate sequences of interesting NMEC plasmids, compare them to other plasmids, determine their phylogeny and evolution, and define their core and pan-genome. Also, we will identify core plasmid genes contributing to NMEC meningitis and their mechanisms of pathogenicity. Remarkably, such an analysis of human ExPEC plasmids has not previously been reported; thus, the work we propose is novel. It is also novel in that it seeks insight into the phylogeny and evolution of NMEC plasmids and their co-evolution with the NMEC chromosome. We believe that this research will make possible many future studies to broaden our understanding of NMEC pathogenicity, the evolution of bacterial virulence, and the role of plasmids in virulence and host adaptation and will lead to new approaches to decrease the occurrence and mitigate the impact of Gram-negative neonatal bacterial meningitis. PUBLIC HEALTH RELEVANCE: Neonatal meningitis-causing Escherichia coli (NMEC) infections result in high morbidity and mortality with survivors experiencing long-term disability. Here, we study one of NMEC's most overlooked putative virulence traits, large pathogenicity island-containing plasmids. Insights gained from this research will lead to better understanding of NMEC pathogenesis and may be used to create more effective meningitis control strategies.